Most animal species are sexually dimorphic, yet the features distinguishing males from females are different in every case. This simple observation implies that new sexual characters are gained, and old ones are lost, during the evolution of any animal lineage. The rapid turnover of sex-specific traits is as obvious in humans and their closest relatives as in other species, but the molecular mechanisms of this turnover are not well understood in any animal group. To address this critical gap in our knowledge, we will use the Drosophila model to identify the genetic changes responsible for the origin of new sexually dimorphic characters. Powerful transgenic technologies allow us to manipulate genome sequence and development in Drosophila in ways that are not possible in any other animal model. Recent work in our lab suggests that the origin of novel sex-specific traits is linked to evolutionary changes in the spatial regulation of doublesex (dsx), a transcription factor that controls sexual differentiation in most somatic tissues. This hypothesis represents a major departure from the previously accepted models, which ascribed the evolution of sexual dimorphism to changes in the target genes regulated by dsx. In this project, we will use a combination of several recently developed transgenic techniques to carry out a rigorous experimental test of the new model. We will focus on the sex comb - a strictly male-specific array of modified sensory organs that evolved recently within the genus Drosophila and shows dramatic diversity among closely related species. Our previous work has shown that sex comb development requires localized expression of dsx and sexually dimorphic expression of the homeotic gene Scr. In species that primitively lack sex combs, dsx expression is absent in the corresponding tissue while Scr expression is sexually monomorphic, suggesting that the origin of sex combs was caused by changes in dsx and Scr regulation. We have identified the DNA sequences (called CREs) that control dsx and Scr expression, and will now characterize the functional impact of the evolutionary changes in these sequences. First, we will compare the regulatory activities of CREs from species with different sex comb morphology and species that lack sex combs, and test whether the origin of the sex comb coincided with the origin of new CREs that drive gene expression in the cells that give rise to this structure. Second, we will replace the dsx and Scr CREs in D. melanogaster with homologous DNA sequences from species with different sex comb morphology and species that lack sex combs, and test whether these replacements are sufficient to eliminate the sex comb or change its appearance. Finally, we will extend this work to other sex-specific structures that evolved independently in distantly related species in order to test the generality of the new model of evolutionary change. A direct experimental confirmation of this model will help explain the origin of sexual dimorphism in all animals.